Triglycerides are one of the major energy storage molecules in eukaryotes. The absorption of triglycerides (also called triacylglycerols) from food is a very efficient process which occurs by a series of steps wherein the dietary triacylglycerols are hydrolyzed in the intestinal lumen and then resynthesized within enterocytes. The resynthesis of triacylglycerols can occur via the monoacylglycerol pathway which commences with monoacylglycerol acyltransferase (MGAT) catalyzing the synthesis of diacylglycerol from monoacylglycerol and fatty actyl-CoA. An alternative synthesis of diacylglycerols is provided by the glycerol-phosphate pathway which describes the coupling of two molecules of fatty acyl-CoA to glycerol-3-phosphate. In either case, diacylglycerol is then acylated with another molecule of fatty acyl-CoA in a reaction catalyzed by one of two diacylglycerol acyltransferase enzymes to form the triglyceride (Farese et al., Curr. Opin. Lipidol., 2000, 11, 229-234).
The reaction catalyzed by diacylglycerol acyltransferase is the final and only committed step in triglyceride synthesis. As such, diacylglycerol acyltransferase is involved in intestinal fat absorption, lipoprotein assembly, regulating plasma triglyceride concentrations, and fat storage in adipocytes. The first diacylglycerol acyltransferase, diacylglycerol transferase 1, was identified in 1960 and the human and mouse genes encoding this protein were isolated in 1998 (Cases et al., Proc. Natl. Acad. Sci. U.S.A., 1998, 95, 13018-13023; Oelkers et al., J. Biol. Chem., 1998, 273, 26765-26771). Mice lacking diacylglycerol acyltransferase 1 are viable and can still synthesize triglycerides through other biological routes, suggesting the existence of multiple mechanisms for triglyceride synthesis (Smith et al., Nat. Genet., 2000, 25, 87-90).
A second diacylglycerol transferase, diacylglycerol transferase 2 (also known as DGAT2, diacylglycerol O-transferase 2, acyl-CoA:diacylglycerol acyltransferase 2), was subsequently identified in the fungus Mortierella, humans and mice (Cases et al., J. Biol. Chem., 2001, 276, 38870-38876; Lardizabal et al., J. Biol. Chem., 2001, 276, 38862-38869). Enzymatic assays indicate that this recently identified protein does possess diacylglycerol transferase activity that utilizes a broad range of long chain fatty acyl-CoA substrates (Cases et al., J. Biol. Chem., 2001, 276, 38870-38876).
Diacylglycerol transferase 2 is a member of a family of genes whose sequences are unrelated to diacylglycerol transferase 1. In addition to differing in sequence compared to diacylglycerol transferase 1, in vitro assays illustrate that diacylglycerol transferase 2 has higher activity at lower concentrations of magnesium chloride and oleoyl-CoA (Cases et al., J. Biol. Chem., 2001, 276, 38870-38876). The predicted protein sequence of diacylglycerol transferase 2 contains at least one putative transmembrane domain, three potential N-linked glycosylation sites, six potential protein kinase C phosphorylation consensus sites, as well as sequences in common with a putative glycerol phosphorylation site found in acyltransferase enzymes (Cases et al., J. Biol. Chem., 2001, 276, 38870-38876). The International Radiation Hybrid Mapping Consortium has mapped human diacylglycerol transferase 2 to chromosome 11q13.3.
In human tissues, the highest levels of diacylglycerol transferase 2 are detected in liver and white adipose tissues, with lower levels found in mammary gland, testis and peripheral blood leukocytes (Cases et al., J. Biol. Chem., 2001, 276, 38870-38876). Two mRNA species of 2.4 and 1.8 kilobases are detected in human tissues, whereas the major diacylglycerol transferase 2 mRNA species in mouse tissues is 2.4 kilobases. In addition to liver and white adipose tissues, diacylglycerol transferase 2 is expressed in all segments of the small intestine in mice, with higher expression in the proximal intestine and lower expression in the distal intestine (Cases et al., J. Biol. Chem., 2001, 276, 38870-38876).
Diacylglycerol transferase activity exhibits distinct patterns during postnatal development of the rat liver. As there is no correlation between the mRNA expression and activity patterns, post-translational modifications may participate in the regulation of diacylglycerol transferase 2 activity during rat development (Waterman et al., J. Lipid. Res., 2002, 43, 1555-1562).
Diacylglycerol transferase 2 mRNA is preferentially upregulated by insulin treatment, as shown by in vitro assays measuring the diacylglycerol activity from the membrane fraction of cultured mouse adipocytes (Meegalla et al., Biochem. Biophys. Res. Commun., 2002, 298, 317-323). In fasting mice, diacylglycerol transferase 2 expression is greatly reduced, and dramatically increases upon refeeding. The expression patterns of two enzymes that participate in fatty acid synthesis, acetyl-CoA carboxylase and fatty acid synthase, respond to fasting and refeeding in a similar fashion. These results, combined with the observation that diacylglycerol transferase 2 is abundantly expressed in liver, suggest that diacylglycerol transferase 2 is tightly linked to the endogenous fatty acid synthesis pathway (Meegalla et al., Biochem. Biophys. Res. Commun., 2002, 298, 317-323).
Studies of mice harboring a disruption in the diacylglycerol acyltransferase 1 gene provide evidence that diacylglycerol acyltransferase 2 contributes to triglyceride synthesis. Levels of diacylglycerol transferase 2 mRNA expression are similar in intestinal segments from both wild type and diacylglycerol transferase 1-deficient mice (Buhman et al., J. Biol. Chem., 2002, 277, 25474-25479). Using magnesium chloride to distinguish between diacylglycerol transferase 1 and 2 activity, Buhman, et al. observed that, in diacylglycerol transferase 1-deficient mice, diacylglycerol transferase activity is reduced to 50% in the proximal intestine and to 10-15% in the distal intestine (Buhman et al., J. Biol. Chem., 2002, 277, 25474-25479).
Additionally, diacylglycerol transferase 2 mRNA levels are not up-regulated the liver or adipose tissues of diacylglycerol transferase 1-deficient mice, even after weeks of high-fat diet (Cases et al., J. Biol. Chem., 2001, 276, 38870-38876; Chen et al., J. Clin. Invest., 2002, 109, 1049-1055). However, in ob/ob mice, which have a mutation in the leptin gene that results in obesity, diacylglycerol transferase 2 is more highly expressed than in wild type mice, suggesting that diacylglycerol transferase 2 may be partly responsible for the highly accumulated fat mass seen in these mice. Furthermore, the combined mutations of leptin and diacylglycerol transferase 1 leads to a three-fold elevation in diacylglycerol transferase 2 expression in white adipose tissue, compared to the levels in the same tissue from diacylglycerol transferase 1-deficient mice (Chen et al., J. Clin. Invest., 2002, 109, 1049-1055). Diacylglycerol transferase 2 mRNA is also upregulated in the skin of these mice (Chen et al., J. Clin. Invest., 2002, 109, 175-181). These data suggest leptin normally downregulates diacylglycerol transferase 2 expression, and that the upregulation of diacylglycerol transferase 2 in white adipose tissue in these mice may provide an alternate pathway for the triglyceride synthesis that still occurs in leptin deficient/diacylglycerol transferase 1-deficient mice (Chen et al., J. Clin. Invest., 2002, 109, 1049-1055).
Diacylglycerol acyltransferase 1 knockout mice exhibit interesting phenotypes in that they are lean, resistant to diet-induce obesity, have decreased levels of tissue triglycerides and increased sensitivity to insulin and leptin (Chen et al., J. Clin. Invest., 2002, 109, 1049-1055; Smith et al., Nat. Genet., 2000, 25, 87-90). As diacylglycerol transferase 2 also participates in triglyceride synthesis, interfering with diacylglycerol transferase 2 may similarly lead to reduced body fat content.
The US pre-grant publications 20030124126 and 20020119138 claim and disclose a nucleic acid molecule encoding human diacylglycerol transferase 2 alpha, as well as compositions, including antisense oligonucleotides, for modulating the activity of said diacylglycerol transferase 2 alpha (Cases et al., 2003).
The US pre-grant publication 20030104414 discloses and claims nucleic acid sequences which are members of a group of genes referred to as “protein cluster V” as well as the method for identification of an agent capable of modulating nucleic acid molecules in the protein cluster V group. This application also discloses the use of RNA interference or double-stranded RNA to disrupt the function of protein cluster V gene family members (Attersand, 2003).
The US pre-grant publication 20030100480 discloses that diacylcglycerol transferase activity, including that of diacylglycerol transferase 2, may be modified by a variety of methods, including antisense, RNA interference and diacylglycerol transferase 2 antisense plasmid constructs (Smith et al., 2003).
The US pre-grant publication 20030028923 claims and discloses a method for modifying the triacylglyerol composition in a plant cell, comprising transforming a plant cell with a nucleic acid construct encoding an enzyme active in the formation of triacylglycerol from diacylglycerol and fatty acyl substrates, including nucleic acid constructs in the antisense orientation. Also disclosed and claimed is a method for ameliorating a disease or condition associated with altered diacylglycerol acyltransferase activity by administering to a subject a therapeutically effective amount of a diacylglycerol acyltransferase agonist. This application discloses that such antagonists can include antisense molecules (Lardizabal et al., 2003).
The PCT publication WO 00/78961 claims and discloses isolated nucleic acid molecules selected from a group including a nucleic acid sequence encoding diacylglycerol acyltransferase 2. This publication also discloses that sense or antisense oligonucleotides binding to target nucleic acid sequences can interfere with transcription or translation of the disclosed and claimed nucleic acid molecules (Baker et al., 2000).
Disclosed and claimed in the PCT publication WO 01/77389 are polynucleotides selected from a group of sequences including a nucleotide sequence encoding a human diacylglycerol acyltransferase. Also claimed and disclosed are a method for screening for the altered expression of said polynucleotides and a method for screening a library of molecules that specifically bind to said polynucleotide sequences (Shiffman et al., 2001).
The PCT publication WO 01/68848 discloses and claims a nucleic acid molecules encoding secreted and transmembrane polypeptides, including a human diacylglycerol acyltransferase 2 nucleic acid molecule, and oligonucleotide probes derived from any of these sequences (Baker et al., 2001).
Disclosed and claimed in the European patent application EP 1 308 459 are a group of polynucleotide sequences, including a nucleic acid molecule encoding human diacylglycerol acyltransferase 2, and antisense polynucleotides against this group of polynucleotide sequences (Isogai et al., 2003).
The PCT publication WO 02/08260 discloses and claims an isolated, purified polynucleotide sequence with identity to a human diacylglycerol transferase 2 nucleotide sequence. This application also discloses a substantially purified oligonucleotide that includes a region of nucleotide sequence that hybridizes to at least 8 consecutive nucleotides of sense or antisense sequence of a nucleotide sequence selected from a group consisting of sequences with identity to human diacylglycerol acyltransferase 2 (Botstein et al., 2002).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of diacylglycerol acyltransferase 2. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting diacylglycerol acyltransferase 1 function.
Antisense technology is an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of diacylglycerol acyltransferase 2 expression.
The present invention provides compositions and methods for modulating diacylglycerol acyltransferase 2 expression.